﻿A new cryptic species of terrestrial breeding frog of the Pristimantisdanae Group (Anura, Strabomantidae) from montane forests in Ayacucho, Peru

﻿Abstract Based on morphological and molecular characters, we describe a new species of terrestrial breeding frog of the Pristimantisdanae Group from montane forests of La Mar Province, Ayacucho Department in southern Peru, at elevations from 1200 to 2000 m a.s.l. The phylogenetic analysis, based on concatenated sequences of gene fragments of 16S rRNA, RAG1, COI and TYR suggests that the new species is a sister taxon of a clade that includes one undescribed species of Pristimantis from Cusco, Pristimantispharangobates and Pristimantisrhabdolaemus. The new species is most similar to P.rhabdolaemus, which differs by lacking scapular tubercules and by its smaller size (17.0–18.6 mm in males [n = 5], 20.8–25.2 mm in females [n = 5] in the new species vs. 22.8–26.3 mm in males [n = 19], 26.0–31.9 mm in females [n = 30] of P.rhabdolaemus). Additionally, we report the prevalence of Batrachochytriumdendrobatidis (Bd) in this species.


Introduction
Pristimantis (Terrarana, Strabomantidae) is an amphibian genus that comprises more than 600 species and occurs thoughout the Americas (Hedges et al. 2008;Padial and De La Riva 2009;Padial et al. 2014;Waddell et al. 2018) from Honduras to Argentina.In Peru, there are 145 Pristimantis, which represents more than 20% of its global richness (Frost 2023).Many species of Pristimantis are morphologically similar despite belonging to different lineages (Elmer and Cannatella 2008;Padial and De la Riva 2009;Siqueira et al. 2009;Kieswetter and Schneider 2013;Hutter and Guayasamin 2015;Ortega-Andrade et al. 2015).The ubiquity of cryptic species in Pristimantis has led to underestimation of the real number of species in the genus (Ortega-Andrade et al. 2015;Guayasamin et al. 2017;Paez and Ron 2019;Carrion-Olmedo and Ron 2021).Nevertheless, the application of molecular techniques in an integrative framework (Dayrat 2005) generated a steady increase in species richness of Pristimantis (Köhler et al. 2022;Reyes-Puig and Mancero 2022).Integrative taxonomy uses more than one line of evidence and discipline to infer species limits (Simpson 1961;Wiley 1978;De Queiroz 2005;Schlick-Steiner et al. 2010;Aguilar et al. 2013;Hutter and Guayasamin 2015) and has become a critical tool to identify and delimit species as well as in understanding species formation (Aguilar et al. 2013;Minoli et al 2014;Rojas et al. 2018;Hillis 2019;Zozaya et al. 2019).
One group with cryptic species includes Pristimantis rhabdolaemus.The taxonomic history is complex because Lynch and McDiarmid (1987) synonymised Pristimantis pharangobates with P. rhabdolaemus, until Lehr (2007) again split these two cryptic species.Incorrect labelling of GenBank sequence EF493706 (uploaded during the period from 1987 to 2007 when synonymy was in place) of P. pharangobates as "P.rhabdolaemus" (Heinicke et al. 2007;Padial et al. 2014;Lehr et al. 2017a, b;Acevedo et al. 2020) contributed to taxonomic confusion.Furthermore, specimens from Bolivia incorrectly assigned to P. rhabdolaemus added more confusion.Despite this history, P. rhabdolaemus species limits have not been studied using integrative taxonomy.
Therefore, as part of a study of Pristimantis rhabdolaemus species boundaries, we collected direct development frogs from the montane forests of La Mar Province, Ayacucho Department.Molecular and morphological analyses revealed that the collected specimens belong to an undescribed species.Our goals are to describe the new species and infer its relationships with other species of the Pristimantis danae species Group, as well as provide information about infection by the fungus Batrachochytrium dendrobatidis (Bd).

Fieldwork and voucher specimens
VHA conducted field research in the montane forest of two small valleys (Fig. 1) in the VRAEM (Spanish acronym for Valley of the Rivers Apurimac, Ene and Mantaro), Ayacucho Department, southern Peru.The fieldwork was organised in two stages.The first occurred from November 2018 to June 2019 in the valley of the Chunchubamba River, which goes from Chiquintirca (2900 m a.s.l.)

Morphology and morphometry
We follow Lynch and Duellman (1997) for the description format, except that we use "vomerine odontophores" instead of "dentigerous processes of vomers" (Duellman et al. 2006) and Duellman and Lehr (2009) for diagnostic characters.We sexed specimens by examining for the presence of vocal slits in mature males and by inspecting gonads in dissected specimens.VHA measured the following distances to the nearest 0.1 mm with digital calipers under a stereomicroscope: 1) snout-vent length (SVL), 2) tibia length (TL, distance from the knee to the distal end of the tibia), 3) foot length (FL, distance from the proximal margin of inner metatarsal tubercle to tip of Toe IV), 4) head length (HL, from the angle of the jaw to tip of snout), 5) head width (HW, at the level of the angle of the jaw), 6) horizontal eye diameter (ED), 7) horizontal tympanum diameter (TY), 8) interorbital distance (IOD), 9) upper eyelid width (EW), 10) internarial distance (IND), 11) eye-nostril distance (EN, straight line distance between anterior corner of orbit and posterior margin of external nares) and one extra measurement: 12) Finger IV disc width (F4).Fingers and toes are numbered pre-axially to postaxial from I-IV and I-V, respectively.We compared the lengths of toes III and V by adpressing both toes against Toe IV; lengths of fingers I and II were compared by adpressing the fingers against each other.Vladimir Díaz Vargas photographed live specimens and VHA preserved the specimens.We used photographs for descriptions of skin texture and colouration.Specimens examined are listed in Appendix 1; other collection acronyms are MUSM = Museo de Historia Natural San Marcos (Lima, Peru); KU = University of Kansas, Museum of Natural History (Kansas, USA); LSUMZ = Louisiana State University Museum of Zoology (Louisiana, USA).

Molecular phylogenetic analysis
We performed a phylogenetic analysis to infer relationships of the new species with other species of the Pristimantis danae Group (Padial et al. 2014).We used fragments of 16S rRNA, COI, RAG1 and TYR genes.We obtained novel sequences by extracting DNA from six specimens of the new species (MUSM 41030, 41031, 41035, 41036, 41037, 41323) with a commercial extraction kit (IBI Scientific, Iowa, USA).We followed Hedges et al. (2008) and von May et al. (2017) for primers and PCR thermocycling conditions.Primers are listed in Table 1.We purified PCR products using Exosap-IT Express (Affymetrix, Santa Clara, CA, USA).MCLAB (San Francisco, CA) performed Sanger sequencing.
We follow Padial et al. (2014) and Pyron and Wiens (2011) for species group assignment within Pristimantis and the choice of outgroup taxa.We downloaded from GenBank all available sequences of 16S rRNA, COI, RAG1 and TYR of other species of the P. danae Group and some of the outgroup taxa.We used selected species of the P. conspicillatus Group (P.bipunctatus, P. iiap and P. skydmainos) and P. prolatus as outgroup taxa (Appendix 2).
We used Geneious Prime version 2023.0.1 (Biomatters, http://www.geneious.com/)to assemble pair-end reads, generate a consensus sequence for each gene and align our novel and GenBank sequences with MAFFT included in Geneious as a plug-in.Posteriorly, we concatenated the four genes (16S, COI, RAG1 and TYR) using the default settings in Geneious.We trimmed aligned  (Lanfear et al. 2017).We inferred a Maximum Likelihood phylogenetic tree withIqTree (Nguyen et al. 2015) by using its web server (http://iqtree.cibiv.univie.ac.at/) with the following settings: ultrafast bootstrap method, 1000 bootstrap alignments and nucleotide evolution models of GTR+I+G for the gene 16S and for 1 st codon position of COI; GTR+G for RAG1, TYR and 3 rd codon position of COI; and GTR for 2 nd codon positions of COI.Additionally, we generated a tree using Bayesian Inference using the plugin MrBayes for Geneious Prime with 1.1 × 10 6 generations and sampling every 200 generations from the Markov Chain Monte Carlo (MCMC).We determined stationarity by plotting the log-likelihood scores of sample points against generation time; when the values reached a stable equilibrium and split frequencies fell below 0.01, stationarity was assumed.We discarded 100,000 samples and 10% of the trees as burn-in.We consider Bayesian Posterior Probabilities (BPP) > 95% as evidence of support for a clade (Huelsenbeck and Ronquist 2001;Wilcox et al. 2002;Aguilar et al. 2013).We visualised both trees in .Finally, we compare uncorrected p-distances of 591 bp (including gaps) of 16S mithocondrial rRNA gene of Pristimantis included in our analysis (included as a separated file in: doi: 10.5281/zenodo.8278333).To estimate p-uncorrected genetic distances, we used MEGA 11 (Tamura et al. 2021) with a variance estimation method of 1000 bootstrap and rates amongst sites of G+I.We excluded from this analysis species from the sister clade (P.bounides, P. aniptopalmatus, P. albertus, P. attenboroughi, P. humboldti, P. danae, P. ornatus, P. puipui, P. reichlei and P. sagittulus, Fig. 2), except from Pristimantis sp.3 from Bolivia because these specimens had been identified as P. rhabdolaemus on GenBank and P. scitulus, because they are novel sequences for this species.

Quantitative PCR assays for fungal infection (Bd)
During fieldwork in 2018, 2019 and 2021, we swabbed live frogs of the new species with a synthetic dry swab (Medical Wire & Equipment #113) to quantify infection by Batrachochytrium dendrobatidis (Bd).We stroked swabs across the skin of juveniles and adults a total of 30 times per individual: five strokes on each side of the abdominal mid-line, five strokes on the inner thighs of each hind leg and five strokes on the foot webbing of each hind leg.We used a standard quantitative Polymerase Chain Reaction (qPCR) assay using DNA extracted from swabs to quantify the level of infection (Boyle et al. 2004).Following the protocol of Boyle et al. (2004) and Hyatt et al. (2007), we extracted DNA from swabs using 40 µl of PrepMan Ultra (Applied Biosystems).We analysed each extract once with a QuantStudio 3 qPCR system (ThermoFisher Scientific).We calculated the number of zoospore equivalents (ZE) (i.e. the genomic equivalent for Bd zoospores) by comparing the sample results with a serial dilution of standards (gBlock synthetic standards, IDT DNA, Iowa, USA).We considered any sample with ZE > 1 to be infected or Bd-positive.

Nomenclatural acts
The electronic version of this article in Portable Document Format (PDF) will represent a published work according to the International Commission on Zoological Nomenclature (ICZ) and, hence, the new name contained in the electronic version is effectively published under that Code from the electronic edition alone.This published work and its nomenclatural acts have been registered in ZooBank, the online registration system for the ICZN.The ZooBank LSIDs (Life Science Identifiers) can be resolved and the associated information is viewed through any standard web browser by appending the LSID to the prefix http://zoobank.org/.The LSID for this publication is urn: urn:lsid:zoobank.org:pub:226A24AB-B4BE-4EFD-BF11-D6CA719AB601.

Molecular phylogenetic analysis
Our Maximum Likelihood phylogeny, based on four concatenated gene fragments (Fig. 2 and Appendix 3 -expanded ML tree), found the new species (in red) included in a clade with specimens of Pristimantis rhabdolaemus, P. pharangobates and P. toftae from their respective type localities, as well as a candidate species from Cusco (in blue).The results from the Bayesian phylogeny (Appendix 4) are largely congruent with the results from the ML phylogeny.
Pristimantis scitulus is within the danae Group and sister to a clade consisting of P. aniptopalmatus, P. humboldti and P. bounides, but with low support.Both our ML and BI phylogenies recover P. danae as paraphyletic, with individuals from the type locality in Cosñipata (Cusco, Peru) forming part of a clade that includes P. danae specimens from Bolivia and P. reichlei, albeit with low support.

Fungal infection by Batrachochytrium dendrobatidis (Bd)
We found six out of 18 specimens of P. similaris (30%) infected by the fungus Batrachochytrium dendrobatidis (Bd), with levels of infection varying from 11.5 to 8889.3 zoospore genomic equivalents.Our finding confirms that species of Pristimantis can be infected with the fungus (Catenazzi et al. 2011;Warne et al. 2016), despite their life cycle excluding aquatic stages and, thus, limiting the frogs' exposure to the aquatic zoospores of Bd.

Species description
Our phylogenetic tree shows a candidate species from Ayacucho with high support and having high genetic distances with closely-related phylogenetic species (see Fig. 2 and Appendix 3).In addition, after a careful examination of its morphology and pattern of colouration, this candidate species shows differences with other species of the P. danae Group.Based on these results, we describe this candidate species from Ayacucho Department as a new species of Pristimantis.Pristimantis similaris sp.nov.https://zoobank.org/BC56FD8A-6EBD-43C9-A446-689FC3253576Figs 4-6 Common name.English: Similar Rubber Frog.Spanish: Rana cutín similar.
Generic placement.We assign this species to the genus Pristimantis, based on morphology and molecular data (Figs 2,4,6).
Diagnosis.A new species of Pristimantis assigned to the P. danae species Group having the following combination of characters: (1) Skin on dorsum shagreen, skin on venter areolate; discoidal and dorsolateral folds present, weak; thoracic fold present; (2) tympanic membrane and tympanic annulus present, distinct, visible externally; (3) snout subaccuminated in dorsal view, round in lateral view; (4) upper eyelid lacking tubercles; EW smaller than IOD; cranial crest absent; two small and flat tubercles above the snout near the eyes; (5) dentigerous processes of vomers low, oblique in five of the paratypes, absent in four paratypes and the holotype; (6) males with vocal slits, subgular vocal sac large extending on to chest and without nuptial pads; (7) Finger I slightly shorter than Finger II; discs of digits expanded, flat and truncated; (8) fingers without lateral fringes; (9) ulnar tubercles present, but diffuse; (10) heel with two to three small and flat tubercles; inner tarsal fold present, small; (11) inner metatarsal tubercle ovoid, 2-3 times larger than outer one; outer metatarsal tubercle small, ovoid; numerous and flat supernumerary tubercles; (12) toes without lateral fringes; basal toe webbing absent; toe V is slightly longer than toe III; toe discs about as large as those on fingers; (13) in life, dorsum varies from blackish to dark brown with three conspicuous chevrons (not very visible in some cases) (Fig. 6); in most of the adults, the anterior surfaces of thighs reddish-orange, posterior surfaces of thighs brown; flanks cream without tubercles; groin same pattern as flanks mostly, some specimens with orange-reddish colouration (Figs 4,6); venter cream to yellow with black conspicuous reticulations in the throat and black marks in the chest, males present yellow throat with black or white longitudinal reticulations not as conspicuous as in females (Figs 4,6); iris dark copper-coloured with fine black vermiculations; (14) SVL in adult females 20.8-25.2mm (mean = 23.4 ± 1.8 SE, n = 5), in adult males 17.0-18.6mm (mean = 18.1 mm ± 0.7 SE, n = 5).
Comparisons.Pristimantis similaris is distinguished from its congeners in Peru and Bolivia by the following combination of characters: skin on dorsum areolate, tympanum and tympanic annulus distinct, weakly-defined discoidal and dorsolateral folds, two small and flat tubercles above the snout near the eyes (not conspicuous in preservative), dorsum dark brown with three darker chevrons, anterior surface of thighs usually orange-reddish and posterior surface of thighs dark brown.Pristimantis similaris can be distinguished from P. rhabdolaemus and P. pharangobates by the following characters (characters in parenthesis): smaller SVL of 20.8-25.8mm in ten females and 15.2-18.9mm in eight males (P.pharangobates 23.1-27.8mm in females and 15.2-18.2mm in males; P. rhabdolaemus 25.5-31.9mm in females and 24.1-26.3mm in males); absence of scapular tubercles (present in both species); presence of conspicuous longitudinal black and white or yellow marks on the throat and chest (less conspicuous in P. pharangobates and P. rhabdolaemus).
Other species in the Pristimantis danae species Group that are similar to P. similaris include P. danae, P. reichlei, P. scitulus and P. toftae.Pristimantis danae and Pristimantis reichlei also have brown chevrons in the dorsum and differ from the new species by the combination of the following characters: males nuptial pads absent (present in P. danae and P. reichlei), dorsolateral folds present (weak in P. danae and absent P. reichlei), small pale spots in the posterior surfaces of the thighs absent (present in P. danae and P. reichlei) and smaller size in P. similaris.Pristimantis scitulus is morphologically similar to P. similaris and has a parapatric distribution (Yuraccyacu, in the Piene Valley, Ayacucho Region).Pristimantis similaris can be distinguished by lacking a conical tubercle in the upper eyelid and heels (present in P. scitulus), mid-ventral line absent (present in P. scitulus) and absence of marks in the groin or thighs (conspicuous dark spots in the groin that is continuous as marks on the posterior surfaces of the thighs in P. scitulus).Pristimantis toftae is a smaller species that is superficially similar to P. similaris.The new species can be distinghished by the absence of coloured marks or spots in the groin or other parts of its body (yellow spot in the groins of P. toftae), absence of labial bar (presence of a white labial bar in P. toftae).
Pristimantis similaris is also similar to some species in the Pristimantis conspicillatus species Group, which includes P. bipunctatus, P. skydmainos and P. iiap.The parapatric Pristimantis bipunctatus (found in Calicanto at 1940 m. a.s.l. in the Piene Valley, Ayacucho Region), has dorsum and ventral skin shagreen and areolate, snout long, upper eyelids without tubercules similar to P. similaris, but the latter differs by having finger I slightly shorter than finger II (finger I and finger II about equal length in P. bipunctatus), discs on outer fingers truncated (broadly rounded in P. bipunctatus), scapular tubercules absent (present in P. bipunctatus) and by its smaller size (22.6-28.8mm in males and 32.4-41.5 mm in females in P. bipunctatus).P. similaris can be distinguished from P. skydmainos by the absence of a mid-dorsal tubercle (present in P. skydmainos), absence of nuptial pads (present in P. skydmainos), finger I smaller than finger II (finger I longer than finger II in P. skydmainos), absence of spots or marks in the posterior surfaces of the thighs (minute cream flecks on the posterior surfaces of the thighs in P. skydmainos) and the absence of W-shaped marks (present in the scapular region in P. skydmainos).Pristimantis similaris differs from P. iiap from the lowland Amazon of the Ucayali Region by lacking large granules on flanks (present in P. iiap), lacking granules on the upper eyelids (present in P. iiap) and by having finger I shorter than finger II (finger I and II about the same length in P. iiap).
Another species with some resemblance (mainly on the throat in ventral view) to the new species is Pristimantis tanyrhynchus.Pristimantis similaris can be distinguished from P. tanyrhynchus by the absence of nuptial pads in males (present in P. tanyrhynchus) and absence of tubercles on the heel (heel with a conical and large tubercle in P. tanyrhynchus).
Description of the holotype.Head longer than wide; head length 43% of SVL; head width 35% of SVL; cranial crests absent; snout subaccuminated in dorsal view and in lateral view (Fig. 4A, B, D); eye-nostril distance same as the eye diameter; nostrils slightly protuberant, directed dorsolaterally; canthus rostralis long, straight in lateral and in dorsal views; loreal region concave; upper eyelid without tubercles, width 90% of IOD (see photo in life Fig.4); supratympanic fold short and narrow, extending from posterior margin of upper eyelid slightly curved to insertion of arm; tympanic membrane and annulus present; distinct conical postrictal tubercles present bilaterally.Choanae small, ovoid, not concealed by palatal shelf of maxilla; dentigerous processes of vomers absent; vocal slits present; tongue longer than short, oval, about a quarter times as long as wide, notched posteriorly, half of the tongue posteriorly free; one large vocal sac extending on to chest.
Skin on dorsum and flanks shagreen, continuous dorsolateral folds present extending from posterior level of tympanic area to level of hind limb insertion; skin on throat, chest and belly areolate; discoidal fold present; thoracic fold present.Outer ulnar surface without tubercles; palmar tubercle bifid; thenar tubercle ovoid; subarticular tubercles well defined, most prominent on base of fingers, ovoid in ventral view, subconical in lateral view; supernumerary tubercles indistinct; fingers long and thin lacking lateral fringes, Finger I shorter than Finger II; tips of digits of fingers expanded, truncated, with circumferential grooves; nuptial pads absent (Fig. 5A).
Hind limbs long, slender, tibia length 58% of SVL; foot length 49% of SVL; dorsal surfaces of hind limbs tuberculate; inner surface of thighs smooth, posterior surfaces of thighs shagreen, ventral surfaces of thighs smooth; heels each with three small conical tubercles; outer surface of tarsus with one minute low tubercle; inner tarsal fold present; inner metatarsal tubercle ovoid, two times the size of round outer metatarsal tubercle; subarticular tubercles well defined, ovoid in ventral view, subconical in lateral view; few plantar supernumerary tubercles, about one quarter the size of subarticular tubercles; toes without lateral fringes; basal webbing absent; tips of digits expanded, truncated, less expanded than those on fingers, with circumferential grooves; relative length of toes: 1 < 2 < 3 < 5 < 4; Toe V slightly longer than Toe III (tip of digit of Toe III and Toe V not reaching distal subarticular tubercle on Toe IV; Fig. 5B).
Colouration of the holotype in life (Fig. 4).In life, dorsum dark brown with three conspicuous chevrons; flanks cream without tubercles, groin same pattern as flanks with orange-reddish colouration; anterior surfaces of thighs reddish-orange, posterior surfaces of thighs brown; venter cream to yellow with black conspicuous reticulations in the throat and black marks in the chest; iris dark copper-coloured with fine black vermiculations (Fig. 4).
Colouration of the holotype in preservative.The dorsal ground colouration is pale brown with three browner chevrons; narrow blackish canthal and supratympanic stripes; flanks pale brown with dark brown and cream flecks forming irregularly-shaped diagonal bars; groin and anterior surfaces of thighs brown with dark brown flecks; chest, belly and ventral surfaces of thighs pale cream, throat pale cream and grey-striped; palmar and plantar surfaces and fingers and toes dark brown; iris pale grey.
Variation.All specimens have the same general appearance, with three chevrons on the dorsum.MUSM 41029 is completely yellow and lacks marks on the chest or throat.MUSM 41032 has two brown-yellowish longitudinal bars on the dorsolateral folds.MUSM 41341 is blackish-brown and the three chevrons are not very visible (Fig. 6).Some individuals (MUSM 41030-2, 41036-7) lack the dentigerous processes of vomers.Morphological measurements ranges and proportions of types are included in Tables 2, 3.
Etymology.The specific name corresponds to the Latin word "similar".This refers to the similarity of the new species and its close phylogenetic relationship with P. rhabdolaemus and P. pharangobates.
Distribution and natural history.The new species is only known from montane forests of Ayna and Anco in Departamento Ayacucho at elevations from 1200-2000 m a.s.l. in secondary forests (Figs 1, 3).This species was found only at night after 18:00 hours, usually perching on wet leaves 0.5-1.5 m above the ground.Males call rarely and their calls are overshadowed by other male species (Pristimantis lacrimosus species Group) calling louder.The species is common and appears to tolerate some human disturbance, because it was found near abandoned farms, less frequented roads and in the sourroudings of abandoned houses.Syntopic species included candidate new species in the Pristimantis lacrimosus species Group and candidate new species in the Pristimantis platydactylus species Group, which were more abundant than the new Table 3. Measurements (in mm) and proportions of adult male and female type specimens of Pristimantis similaris sp.nov.; ranges followed by means and one standard deviation in parentheses.For abbreviations, see Materials and methods.

Discussion
We describe Pristimantis similaris, a new species morphologically similar and phylogenetically related to P. rhabdolaemus and P. pharangobates.Despite their confusing taxonomic history (see Introduction), our phylogenetic analyses show that P. rhabdolaemus and P. pharangobates are distinct evolutionary lineages.
Pristimantis rhabdolaemus was described from mid-altitude montane forests restricted to Ayacucho and Cusco Departments (Duellman 1978).Although we visited the type locality of P. rhabdolaemus (Machente, Ayacucho Department) at 1650 m a.s.l., we could not find any specimens from this species.For that reason, we sequenced specimens of P. rhabdolaemus from Toccate, Anchihuay district in the Ayacucho Department (~ 38 km straight air line to Machente) because morphological analysis and comparison with the type series confirmed that these specimens corresponded to P. rhabdolaemus sensu stricto.Padial et al. (2007) reported P. rhabdolaemus from Bolivia on the basis of 16S rRNA of two incorrectly assigned specimens (MNKA 6628 and MNCN 43036), but our concatenated phylogeny suggests that the Bolivian specimens belong to a different and probably undescribed species, Pristimantis sp. 3 (in purple, Fig. 2).Likewise, P. pharangobates should be restricted to Cusco Department until molecular data become available and support the presence of this species in Puno (south-eastern Peru) and Bolivia.
We also found another candidate species from Cosñipata and Alfamayo in Cusco, morphologically similar and phylogenetically related to P. pharangobates and P. rhabdolaemus (in blue, Fig. 2).Additional specimens and analyses are needed to assess the taxonomic status of these potential new species.
The taxonomy of other species of the P. danae species Group requires further work.For instance, specimens identified as P. danae or P. reichlei tend to form paraphyletic groups in phylogenies.We suggest that both species might benefit from future studies clarifying the phylogenetic relationships of their assigned populations.Such studies might include the use of genomic data for these species (including P. toftae) because the use of four genes (three nuclear) in this study was not sufficiently informative to infer with confidence phylogenetic relationships between the most inclusive clades.
Furthermore, our phylogenies include for the first time sequences of P. scitulus from Chungui, Ayacucho previously known only from two type specimens in Yuraccyacu, Ayacucho (at 2600 m a.s.l) and supports the assignment of this species in the P. danae species Group.We also included sequences for the first time of P. iiap (outgroup) and it is recovered in the P. conspicillatus species Group.
According to Swenson et al. (2012), who discussed the endemism of species (birds, mammals, plants and amphibians) in the eastern Andean slopes from the treeline (~ 3500 m a.s.l.) to the Amazon lowlands, most of the montane forests in the eastern Andean slopes of Peru and Bolivia (fig.7 in Swenson et al. (2012)) are centres of endemism, specially in areas with little field evaluations due to social problems, such as montane forests in Ayacucho Department.
We would like to highlight the areas surroundings the type locality of P. similaris and closely-related species in south-eastern Peru.The Departments of Ayacucho and Cusco have biologically "irreplaceable areas" due to the configuration of the western Andes, the eastern Cordillera de Vilcabamba and the Apurimac River (Swenson et al. 2012).These geographical formations created a deep canyon along the Apurimac River at the border of the Departments of Ayacucho and Cusco (Lehr and Catenazzi 2008;Hazzi et al. 2018;Herrera-Alva et al. 2020), dissecting the Andean cordillera and providing mid-altitude isolated areas.The Apurimac Canyon is an important barrier for the dispersal of amphibians, such as high-altitude species of Terrarana: the Canyon splits the distribution of the genus Phrynopus to the northeast part of the Canyon from the distribution of Bryophryne southwest of the Canyon (Lehr and Catenazzi 2008, 2009, 2010).We believe that this pattern can be extended to mid-altitude montane forest frogs, such as species in the P. danae or P. lacrimosus species groups (pers.com.Ernesto Castillo-Urbina) or the distribution of mid-altitude toads, such as Atelopus moropukaqumir (northwest of the Apurimac Canyon) and A. erythropus (southeast of the Canyon; Herrera-Alva et al. ( 2020)).Therefore, we hypothesise that the Apurimac Canyon could have promoted vicariant speciation of morphologically-similar Pristimantis in these montane forests.For instance, P. similaris occurs from 1200 to 2000 m a.s.l. on the northwest of the Apurimac Canyon in Ayacucho Department, while Pristimantis sp.ocurrs from 1200-2000 m a.s.l. in Cusco Department, southeast of the Apurimac Canyon.Nevertheless, the Apurimac Canyon might have not been a geographic barrier to other species, such as P. rhabdolaemus which has been found at both sides of the canyon.One population of this species has an altitude range from 2000-2900 m a.s.l. in Ayacucho (eastern part of the Apurimac River) and the other population ranges from 2000-2100 m a.s.l. in Cusco (western part of the Apurimac River) according to available specimens and sequences.The presence of P. rhabdolaemus on both sides of the Apurimac River will remain hypothetical until more specimens and tissues from Cusco Department become available and are tested against a hypothesis of two different species following an integrative approach.
We also provide information about infection by the fungus Batrachochytrium dendrobatidis (Bd).Chytridiomycosis, caused by the Bd fungus, has negatively affected amphibian communities in the montane forests of Central America and South America (Berger et al. 1998;Lips et al. 2008;Catenazzi et al. 2011;Catenazzi 2015).This pathogen has been associated with amphibian worldwide declines (Berger et al. 1998;Briggs et al. 2005;Lips et al. 2006;Catenazzi et al. 2010;Scheele et al. 2019).Catenazzi et al. (2011) reported a rapid decline in frog species richness and abundance from 1999 to 2008 in the upper Manu National Park (Cusco), which has communities and ecosystems similar to those found in our study area (Ayacucho).The high prevalence of 30% in P. similaris suggests that Bd could be threatening amphibians in the area and that Bd transmission (which is typically associated with aquatic species, given that the infective zoopores are aquatic) occurs in terrestrial frogs.

Figure 1 .
Figure 1.Montane forest of Cajadela (A) and Machente (B), Ayacucho Department.Type locality of Pristimantis similaris sp.nov. in Cajadela (A).Note the presence of secondary forest in both localities.Photo A taken on 8 November 2021 and B, on 24 October 2022.Arrow marks the type locality.

Figure 3 .
Figure 3. Distribution map of some species of the Pristimantis danae species Group in Ayacucho and Cusco Departments.Raster of altitude from 500 to 4500 m. a.s.l.(white to black).Locality of new species in red circles.

Figure 6 .
Figure 6.A-H colour and pattern variation of Pristimantis similaris sp.nov.Specimen from A-F collected in Cajadela: A, B male MUSM 41029 C, D female MUSM 41031 E, F female MUSM 41032.Specimen G, H male MUSM 41326 collected in Machente.Photos by V. Diaz-Vargas and E. Castillo-Urbina.
Figure A1.Maximum Likelihood tree non-collapsed of concatenated genes 16S rRNA, COI, RAG1 and TYR taken from GenBank and novel sequences.Numbers on nodes are bootstrap values (see Materials and Methods section for details).Green shadow corresponds to the ingroup.Pristimantis similaris sp.nov. in red, Pristimantis sp. 3 from Bolivia in purple and Pristimantis sp. from Cusco in blue.

Table 1 .
Primers employed in this study for PCR and DNA sequencing.F = forward, R = reverse.

Table 2 .
Morphological measurements (mm) of nine paratype specimens of Pristimantis similaris sp.nov.For abbreviations, see Materials and methods.